It has been almost ten years since the first description of germ cell formation from human embryonic stem cells (ESCs). In the intervening years this work has been repeated and extended by multiple groups and now includes the capacity to generate germ line cells from human induced pluripotent stem cells (IPSCs) as well as the generation of haploid cells entirely in vitro. One of the goals of generating germ line cells from human IPSCs is to use the in vitro germ line to treat infertility. However, the capacity to test germ line quality and function is not possible due to the highly experimental nature of this technology. In contrast using mice it has been shown that immature germ line progenitors called primordial germ cells (PGCs) created entirely in vitro from IPSCs can be transplanted into the seminiferous tubules to treat male infertility. Therefore, in this project, our goal is to generate germ line cells in vitro from the primate Rhesus macaque in order to evaluate transplantability and functionality using in vitro fertilization assays. In our fist aim, we will characterize rhesus PGCs at the stage in which transplantation was successfully achieved in mice. In the second aim we will induce rhesus PGC formation in vitro and compare the identity of the in vitro PGCs to the endogenous counterparts. In this aim, we will compare PGC formation from three different types of PSCs namely rhesus ESCs (rESCs), somatic cell nuclear transfer (cloned) rhesus ES (ORES) cells and rhesus IPSC (rIPSCs) generated from isogenic fibroblasts to the ORES cells. This will enable us to evaluate similarities and differences in PGC formation between independently sourced PSC lines. We will also create haploid cells entirely in vitro from rhesus PSCs to test capacity to fertilize rhesus oocytes. In the third aim our goal is to derive autologous riPSC from pre pubescent Rhesus macaques, generate PGCs in vitro and determine whether the in vitro PGCs are able to reconstitute spermatogenesis following autologous transplantation to the seminiferous tubule epithelium. At the conclusion of this project we will know definitively whether in vitro derived primate PGCs have the capacity to reconstitute spermatogenesis in vivo and to fertilize oocytes in vitro to give rise to embryos of the next generation.